Transport of proteins and RNA between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). NPC-mediated transport occurs in both directions through the nuclear envelope. All nuclear proteins are imported from the cytoplasm, their site of synthesis. Transfer RNA (tRNA) and messenger RNA (mRNA) are exported from the nucleus, their site of synthesis, to the cytoplasm, their site of function. Furthermore, the biogenesis of the ribonucleoproteins involves multiple transport steps. Processing of small nuclear RNAs (snRNAs) involves export into the cytoplasm, assembly with Sm proteins and modifications such as hypermethylation to produce small nuclear ribonuclear proteins (snRNPs), and subsequent import of the snRNPs back into the nucleus. The assembly of ribosomes requires the initial import of ribosomal proteins from the cytoplasm, their incorporation with RNA into ribosomal subunits, and export back to the cytoplasm. Gorlich, D. and Mattaj, I. W. (1996; Science 271:1513-1518) estimated that in a HeLa cell which divides every 24 hours, approximately 100 ribosomal proteins and 3 ribosomal subunits must travel through a single NPC per minute.
The transport of proteins and RNPs across the NPC is generally selective and signal-dependent. Nuclear localization sequences (NLSs) are usually characterized by one or more clusters of basic amino acids, but they do not conform to a tight consensus (Gorlich and Mattaj, supra). For instance, the NLS of the large T antigen of simian virus 40 (SV40) was initially found by point mutations which misdirected the protein to the cytoplasm. The SV40 T antigen NLS is a seven amino acid sequence which is sufficient to confer nuclear localization even when conjugated as a synthetic polypeptide to another protein such as serum albumin. Saturation of the protein import pathway by microinjection of high concentrations of NLS-peptide conjugates provided strong evidence for the existence of saturatable NLS receptors. Experimental evidence suggests that there are multiple receptors for different NLS-containing protein substrates (Gorlich and Mattaj, supra).
The NPCs, or nucleoporins, are estimated to contain about 100 different polypeptides. A heterodimeric protein complex consisting of importin alpha and importin beta (also known as karyopherin alpha and beta) targets the NLS-containing substrate proteins to the NPCs. The alpha subunit of importin functions as the NLS receptor, whereas the beta subunit mediates docking to the NPC. The small GTPase Ran mediates the energy-dependent translocation of the substrate-receptor complex through the NPC. After translocation, the importin heterodimer dissociates: importin alpha and the substrate enters and accumulates in the nucleoplasm, and importin beta accumulates at the nuclear pore complex. Ran-GTP induces the dissociation of the importin subunits by forming a complex with importin beta (Gorlich and Mattaj, supra). Multiple importin-alpha homologs have been identified in mouse and in man (Weis, K. et al (1995) Science 268:1049-1053; Seki, T. et al. (1997) Biochem. Biophys. Res. Comm. 234:48-53).
Defective nuclear transport plays a role in cancer. The BRCA1 protein contains three potential NLSs which interact with importin alpha and is transported into the nucleus by the importin/NPC pathway. Recently, Chen, C. F. et al. (1996; J. Biol. Chem. 271:32863-32868) reported that in breast cancer cells the BRCA1 protein is aberrantly localized in the cytoplasm. Wild-type BRCA1 protein expressed in six different breast cancer cell lines was localized in the cytoplasm. However, wild-type BRCA1 protein expressed in four non-breast cancer cell lines was localized in the nucleus. Chen et al. (supra) proposed that the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the importin/NPC nuclear import pathway.
Moll, U. M. et al. (1992; Proc. Natl. Acad. Sci. USA 89:7262-7266) demonstrated that in 10 of 27 cases of breast cancer, p53 tumor suppressor protein was found in the cytoplasm. A majority of the p53 cDNAs derived from breast cancers with cytoplasmic p53 protein were wild type. In contrast, p53 cDNAs derived from breast cancers with nuclear p53 protein contained a variety of missense mutations and a nonsense mutation. Moll et al. (supra) suggested that in some breast cancers, the tumor-suppressing activity of p53 is inactivated by the sequestration of the protein in the cytoplasm, away from its site of action in the cell nucleus. Furthermore, the presence of wild-type p53 in the cytoplasm of normal lactating breast tissue suggested that this mechanism may be employed in specific physiologic situations to permit transient cell proliferation. Cytoplasmic wild-type p53 was also found in human cervical carcinoma cell lines (Liang, X. H. et al. (1993) Oncogene 8:2645-2652).
Nuclear transport by the importin/NPC pathway also plays a role in infection by HIV and other viruses. HIV can infect nondividing cells (such as terminally differentiated macrophages, mucosal dendritic cells, and quiescent T-lymphocytes) because its preintegration complex is recognized by and is actively transported through the NPC. The uncoated HIV-1 preintegration complex contains three proteins, matrix protein MA, Vpr, and integrase IN, known to contain NLSs which interact with importin alpha and which play important roles in the nuclear import and the viral integration processes (Gallay, P. et al. (1997) Proc. Natl. Acad. Sci. USA 94:9825-9830). Furthermore, the NLS-dependent nuclear import activity of the HIV preintegration complex permits the in vivo delivery of transgenes by HIV-derived retroviral vectors into terminally differentiated cells such as neurons. In contrast, oncoretroviruses such as the murine leukemia virus and oncoretroviral vectors cannot traverse an intact nuclear envelope, which precludes their integration into nondividing cells (Gallay et al., supra).
Epstein-Barr virus (EBV) is the etiological agent of infectious mononucleosis and the putative inductive agent of several malignancies. The EBV nuclear antigen-1 (EBNA1), the only EBV protein necessary for replication and maintenance of the EBV genome in the infected cell, interacts with importin alpha through its NLS and is transported into the nucleus by the importin/NPC pathway (Fisher, N. et al. (1997) J. Biol. Chem. 272:3999-4005). Other viral proteins known to contain NLSs include nucleoprotein and the RNA polymerase subunits of influenza virus; VP1, VP2, and VP3 proteins of SV40; and hexon and several coat proteins of adenovirus (Gallay et al., supra).
The discovery of a new human importin alpha homolog and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of disorders associated with cell proliferation, viral infection, and the immune response.